1. Field of the Invention
The present invention relates to a multilayer chemical analytical element employable for determination of a specific substance contained in fluids taken from living body, such as blood. More particularly, the invention relates to a multilayer analytical film suitably employed for the measurement of transaminase activity utilizing the reaction in which pyruvic acid oxidase participates.
2. Description of Prior Art
Various kinds of transaminase are known as amino group-transferring enzymes. Particularly, as for alanine aminotransferase (ALT) and aspattic acid aminotransferase (AST), variation of the concentration thereof in blood is one criterion in finding a liver disease, so that the measurement of ALT or AST activity is very important in the diagnosis of the liver disease.
ALT and AST are enzymes for catalyzing the following reactions (1) and (2), respectively: ##STR1##
As a method for measuring the activity of various transaminases in the living body, there is known a method comprising the steps of producing pyruvic acid by reaction of the transaminase with a substrate, and subjecting the produced pyruvic acid to reaction with pyruvic acid oxidase and a peroxidase-containing color-forming indicator composition for detecting hydrogen peroxide for measuring the amount of the pyruvic acid by colorimetry to determine the transaminase activity, as described in Japanese Patent Provisional Publication No. 55(1980)-13068.
The principle of the method can be illustrated by the following representative reaction formula: ##STR2##
Abbreviations in the above formulae represent the following:
AST: aspartic acid aminotransferase PA1 ALT: alanine aminotransferase; PA1 Pi: inorganic phosphoric acid; PA1 POP: pyruvic acid oxidase; PA1 FAD: flavine adenine dinucleotide PA1 TPP: thiamine pyrophosphate PA1 M.sup.2+ : divalent metal; and PA1 POD: peroxidase.
In more detail, pyruvic acid is produced in the reaction (1) catalyzed by ALT. Otherwise, oxaloacetic acid produced in the reaction (2) catalyzed by AST is converted by the action of oxaloacetic acid dehydrocarbon enzyme (4) to produce pyruvic acid. The pyruvic acid thus produced is converted to hydrogen peroxide according to the corresponding conjugated reaction (3) catalyzed by POP. Through the reaction in which the hydrogen peroxide serves as a substrate, a hydrogen donor and a coupler react with each other a coupling reaction (5) to give a dye. The thus produced dye is determined by colorimetry.
When the above-described determination method is used for an aqueous liquid, a complicated and time consuming series of chemical reactions in the aqueous liquid are required. Further, an interference from so-called intrinsic pyruvic acid (namely, pyruvic acid originally contained in the liquid sample) may present an even more complicated operation and longer period of time are required for the analysis in order to prevent the interference.
For coping with these difficulties in the liquid system, a so-called dry chemistry has been developed. An integral multilayer analytical element employable in the dry chemistry is described, for instance, in Japanese Patent Publication No. 53(1978)-21677 and Japanese Patent Provisional Publication No. 55(1980)-164356. A multilayer analytical element for the measurement of transaminase activity according to the dry chemistry using pyruvic acid oxidase is described in Japanese Patent Provisional Publication No. 57(1982)-144996. Recently, it is desired that the influence by various interfering substances be decreased and the analysis can be performed by a rapid and simple operation. One of the interfering substances is the intrinsic pyruvic acid.
The normal concentration of the pyruvic acid in human blood (e.g., serum, blood plasma and whole blood) generally ranges from 0.3 to 0.6 mg/dl, and the concentration sometimes reaches an abnormally high value of not lower than 2 mg/dl. The concentration of the pyruvic acid in blood increases in the case of diseases, such as, serious cirhosis of liver, liver coma or uremia. There are certain control serums having a high concentration of pyruvic acid ( e.g., not lower than 2 mg/dl ).
In the case of measuring the transaminase activity within a relatively short period of time (e.g., less than 10 minutes) using a conventional multilayer analytical element described for instance in the aforementioned Japanese Patent Provisional Publication No. 57(1982)-144996, an error is sometimes observed in the measurement due to the pyruvic acid contained in the liquid sample, i.e., intrinsic pyruvic acid.